The long-term goal of this 5-year Grant Application is to understand basic issues on the molecular physiology of the thiazide-sensitive Na-CI cotransporter. The Gitleman's disease is the result of inactivating mutations of this gene and this cotransporter is one of the genes that could be implicated in the development of human hypertension. The renal Na-CI cotransporter is the target of the thiazide-type diuretics, which are among the most commonly prescribed drugs in the world. Moreover, the expression of the thiazide-sensitive cotransporter is highly regulated by multiple factors that are known to modulate the renal excretion of sodium. Thus, the renal Na-CI cotransporter is of major importance in renal physiology, pharmacology, and pathophysiology. Using a functional expression strategy in X. laevis oocytes, we have demonstrated important differences between the mammalian and fish thiazide-sensitive Na-CI cotransporter in the specificity and kinetic properties for ion translocation, the diuretic binding affinity and the response to regulation by cell volume and by WNK4 kinase. The major focus of this Application is to determine the domains and/or single amino acid residues defining these functional differences. The specific hypothesis to be examined in this application are that differences in diuretic affinity and binding site are located in the extracellular connecting loops, that differences in ion transport kinetics are located within diverge transmembrane domains and that regulatory differences are located in the intracellular N- and C-terminal domain. Using a combination of molecular, biochemical, and physiological approaches, we will a) identify the diuretic binding site of the renal thiazide-sensitive Na:CI cotransporter; b) identify the structural determinants of ion affinity in the renal thiazide-sensitive Na:CI cotransporter, and c) identify the structural requirements for TSC/NCC regulation by cell volume and by WNK kinases. [unreadable] [unreadable] [unreadable]